Spectral flow cytometry cluster analysis of therapeutic donor lymphocyte infusions identifies T cell subsets associated with outcome in patients with AML relapse

Abstract

Identification of immune phenotypes linked to durable graft-versus-leukemia (GVL) response following donor lymphocyte infusions (DLI) is of high clinical relevance. In this prospective observational study of 13 AML relapse patients receiving therapeutic DLI, we longitudinally investigated changes in differentiation stages and exhaustion markers of T cell subsets using cluster analysis of 30-color spectral flow cytometry during 24 months follow-up. DLI cell products and patient samples after DLI were analyzed and correlated to the clinical outcome. Analysis of DLI cell products revealed heterogeneity in the proportions of naïve and antigen experienced T cells. Cell products containing lower levels of effector memory (eff/m) cells and higher amounts of naïve CD4+ and CD8+ T cells were associated with long-term remission. Furthermore, investigation of patient blood samples early after DLI showed that patients relapsing during the study period, had higher levels of CD4+ eff/m T cells and expressed a mosaic of surface molecules implying an exhausted functional state. Of note, this observation preceded the clinical diagnosis of relapse by five months. On the other hand, patients with continuous remission retained lower levels of exhausted CD4+ eff/m T cells more than four months post DLI. Moreover, lower frequencies of exhausted CD8+ eff/m T cells as well as higher amounts of CD4+temra CD45RO+ T cells were present in this group. These results imply the formation of functional long-term memory pool of T cells. Finally, unbiased sample analysis showed that DLI cell products with low levels of eff/m cells both in CD4+ and CD8+ T cell subpopulations associate with a lower relapse incidence. Additionally, competing risk analysis of patient samples taken early after DLI revealed that patients with high amounts of exhausted CD4+ eff/m T cells in their blood exhibited significantly higher rates of relapse. In conclusion, differentially activated T cell clusters, both in the DLI product and in patients post infusion, were associated with AML relapse after DLI. Our study suggests that differences in DLI cell product composition might influence GVL. In-depth monitoring of T cell dynamics post DLI might increase safety and efficacy of this immunotherapy, while further studies are needed to assess the functionality of T cells found in the DLI.

Full spectrum flow cytometry in the clinical laboratory

“…While there are far too many to list, we refer the reader to several examples of non-clinical assays with human specimens. [20][21][22][23][24][25][26] The above method-comparison studies provide strong preliminary evidence that spectral flow cytometers are likely functionally equivalent to conventional flow cytometers but are superior in the sense that they can perform high-dimensional assays in a single-tube format that otherwise requires 4 separate tubes on current 10-to-12-color conventional flow cytometers. It is critical that comprehensive validation studies be published in reputable peer-reviewed journals with comparisons against regulatory agency-approved predicate conventional flow cytometers.…”

“…In addition to these studies, there are many examples in the research literature where spectral flow cytometry assays have been used successfully to immunophenotype human blood, bone marrow, or lymph nodes. While there are far too many to list, we refer the reader to several examples of non‐clinical assays with human specimens 20–26 …”

Brief research report: in-depth immunophenotyping reveals stability of CD19 CAR T-cells over time

“…Beyond conventional gating, we performed an unsupervised cluster analysis ( 26 ) of the infused CAR T-cell product (n=11) as well as across all available samples (n=29). As described in the Methods section cells were separated into 15 distinct clusters for the analysis of the infused CAR T-cell products ( Figure 1F ) and 20 distinct clusters in the case of all samples analysis ( Supplementary Figure S2A ).…”

The profile and prognostic significance of bone marrow T-cell differentiation subsets in adult AML at diagnosis

“…Adoptive cell transfer (ACT) is one of the major immunotherapeutic strategies in AML. Compared to terminally differentiated T cells, donor lymphocyte infusions (DLI) in relapsed AML patients with higher proportions of early-differentiated memory T cells showed superior durability and anti-tumor activity ( 21 ). In addition, early-differentiated memory T cells expressed higher levels of immune checkpoints compared relapsed to CR AML patients after allo-HSCT ( 22 ).…”

“…In AML, CD8 Tn-derived donor cells effectively combated AML blasts in immunodeficient mice ( 35 ). Donor lymphocyte infusions (DLI) with cell products containing higher CD4 and CD8 Tn proportions were associated with a longer-term remission in AML ( 21 ). Consistently, our study demonstrated that increased BM CD8 Tn at diagnosis independently predicted both favorable RFS and EFS in AML.…”